Our research is focused on elucidating the mechanisms that underlie ATP-dependent protein remodeling carried out by molecular chaperone machines and the role of chaperones in ATP-dependent proteolysis. Molecular chaperones function during non-stress conditions to facilitate folding of newly synthesized proteins, to remodel protein complexes, and to target regulatory proteins and misfolded proteins for degradation. During cell stress, chaperones play an essential role in preventing folding intermediates from becoming irreversibly damaged and forming protein aggregates. They promote recovery from stress by disaggregating and reactivating proteins. They are also involved in delivering damaged proteins to compartmentalized proteases. Protein aggregation, misfolding and premature degradation are major contributors to a large number of human diseases, including cancer. The goal of our research is to understand how chaperones function and to provide the foundation for discovering preventions and treatments for diseases involving protein misfolding. One aim is to understand the mechanism of action of Hsp90. The Hsp90 family of heat shock proteins represents one of the most abundantly expressed and highly conserved families of molecular chaperones. Eukaryotic Hsp90 is known to control the stability and the activity of more than 200 client proteins, including receptors, protein kinases and transcription factors. Hsp90 is also important for the growth and survival of cancer cells and drugs targeting Hsp90 are currently in clinical trials. To gain insight into the mechanism of action of this important family of chaperones, we are studying Hsp90 from Escherichia coli, Hsp90Ec, and from yeast, Hsp82. We discovered that Hsp90Ec, and the Hsp70 chaperone system of E. coli, the DnaK system, act synergistically in protein reactivation in vitro. ATP hydrolysis by Hsp90Ec is required, showing for the first time that Hsp90Ec exhibits ATP-dependent chaperone activity. Our work shows that Hsp90Ec interacts directly with the E. coli Hsp70, DnaK. However, the binding is weak. To explore this interaction, we tested whether binding of a client protein or DnaK cochaperones affects the stability of the Hsp90Ec-DnaK complex. Using an in vitro protein-protein interaction assay, we found that Hsp90Ec formed a significantly more stable ternary complex with DnaK and L2, a client protein known to interact with Hsp90Ec, than with DnaK alone. CbpA, a J-domain protein, promoted further stabilization of Hsp90Ec-DnaK-L2 complex. Additional results using Hsp90Ec and DnaK mutants defective in substrate binding or ATP hydrolysis demonstrated that client binding as well as ATP hydrolysis by both DnaK and Hsp90Ec were necessary for ternary complex formation. To determine the region of Hsp90Ec that interacts with DnaK we developed a screen using a bacterial two-hybrid system to isolate Hsp90Ec mutants potentially defective in DnaK interaction. In vitro these mutants were defective in both functional interaction and physical interaction with DnaK. From this work we concluded that a region of Hsp90Ec in the middle domain of the protein interacts with DnaK. By using molecular docking we identified a probable region in the nucleotide-binding domain of DnaK that interacts with the middle domain of Hsp90Ec. We then made substitution mutants in DnaK residues predicted from the model to interact with Hsp90Ec. Most of the mutants were defective or partially defective in their ability to interact with Hsp90Ec in vivo in a bacterial two-hybrid assay and in vitro in a bio-layer interferometry assay. The mutants were also defective in their ability to function in protein reactivation in combination with DnaK and DnaK cochaperones. The region of DnaK we identified as important for interacting with Hsp90Ec overlaps with the region of DnaK that interacts with J-domain proteins. This work provides a better understanding of the regulation of the chaperone activity of Hsp90Ec by DnaK and offers new insight in improving inhibitor design at specific stages of the Hsp90 chaperone cycle. A second aim is to elucidate the mechanism of protein disaggregation by Clp/Hsp100 molecular chaperones, including ClpB of prokaryotes and its yeast homolog, Hsp104. Understanding how energy-dependent protein disaggregating machines function is clinically relevant since protein aggregation is linked to medical conditions, including Alzheimer's disease, Parkinson's disease, amyloidosis and prion diseases. ClpB/Hsp104 promotes cell survival following stress by disaggregating protein aggregates and reactivating stress-inactivated proteins. ClpB and Hsp104 act with a second molecular chaperone system, DnaK in E. coli and Hsp70 in yeast. To identify the site on E. coli DnaK that interacts with ClpB, we substituted amino acid residues throughout the DnaK NBD. We found that several variants with substitutions in subdomains IB and IIB of the DnaK NBD were defective in ClpB interaction in vivo in a bacterial two-hybrid assay and in vitro in a fluorescence anisotropy assay. The DnaK subdomain IIB mutants were also defective in the ability to disaggregate protein aggregates with ClpB, DnaJ and GrpE, although they retained some ability to reactivate proteins with DnaJ and GrpE in the absence of ClpB. We observed that GrpE, which also interacts with subdomains IB and IIB, inhibited the interaction between ClpB and DnaK in vitro, suggesting competition between ClpB and GrpE for binding DnaK. Computational modeling of the DnaK-ClpB hexamer complex in collaboration with the Stan laboratory (University of Cincinnati) indicated that one DnaK monomer contacts two adjacent ClpB protomers simultaneously. The model and the experimental data support a common and mutually exclusive GrpE and ClpB interaction region on DnaK. Additionally, homologous substitutions in subdomains IB and IIB of Ssa1 caused defects in collaboration between Ssa1 and Hsp104. Altogether, these results provide insight into the molecular mechanism of collaboration between the DnaK/Hsp70 system and ClpB/Hsp104 for protein disaggregation. A third aim is to investigate the mechanism of action of Clp chaperones in proteolysis. Clp proteases of prokaryotes have analogous structures, functions and mechanisms of action to the eukaryotic proteasome. They are composed of an ATP-dependent protein unfolding component and a protease component. ClpXP is a two-component ATP-dependent protease that unfolds and degrades proteins bearing specific recognition signals. Some Clp proteases, including ClpXP, are regulated by adaptor proteins and anti-adaptor proteins. In collaboration with Dr. Susan Gottesman's laboratory (NCI), we are studying how ClpXP is regulated by RssB, an adaptor protein, and IraP, IraD and IraM, three anti-adaptor proteins. RssB specifically targets RpoS, the stationary phase sigma factor of E. coli, for degradation during exponential growth. In response to stress, RpoS degradation ceases. IraP, IraM and IraD, are each made under a different stress condition and interact with RssB to block RpoS degradation. To understand the mechanism of the regulation of ClpXP by adaptors and anti-adaptors, we have been studying in collaboration with the Gottesman group the physical and functional interactions between RssB, ClpX, RpoS and IraP in vitro.